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Protein Folding01:25

Protein Folding

Proteins are chains of amino acids linked together by peptide bonds. Upon synthesis, a protein folds into a three-dimensional conformation, critical to its biological function. Interactions between its constituent amino acids guide protein folding, and hence the protein structure is primarily dependent on its amino acid sequence.
Protein Structure Is Critical to Its Biological Function
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Many proteins form complexes to carry out their functions, making protein-protein interactions (PPIs) essential for an organism's survival. Most PPIs are stabilized by numerous weak noncovalent chemical forces. The physical shape of the interfaces determines the way two proteins interact. Many globular proteins have closely-matching shapes on their surfaces, which form a large number of weak bonds. Additionally, many PPIs occur between two helices or between a surface cleft and a polypeptide...
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The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis
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The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

Published on: March 17, 2010

Peak compression factor of proteins.

Fabrice Gritti1, Georges Guiochon

  • 1Department of Chemistry, University of Tennessee, Knoxville, TN 37996-1600, USA.

Journal of Chromatography. A
|July 17, 2009
PubMed
Summary

This study introduces a method to accurately measure protein band compression in gradient RPLC, finding experimental values are lower than theoretical predictions due to pressure gradients.

Area of Science:

  • Analytical Chemistry
  • Chromatography
  • Biophysical Chemistry

Background:

  • Gradient Retention in Reversed-Phase Liquid Chromatography (RPLC) is crucial for protein separation.
  • Accurate measurement of band compression factor G(12)(2) is essential for optimizing RPLC methods.
  • Extra-column band broadening and mobile phase composition effects on retention and plate height need consideration.

Purpose of the Study:

  • To propose and validate an experimental protocol for precise measurement of the band compression factor G(12)(2) for proteins in gradient RPLC.
  • To investigate the influence of gradient time on the band compression factor.
  • To compare experimental results with theoretical models and identify discrepancies.

Main Methods:

  • Experimental measurement of band compression factor G(12)(2) for insulin using gradient RPLC.

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  • Utilized a C4-bonded BEH-silica column (2.1mm x 50mm, 1.7 µm particles).
  • Employed linear acetonitrile gradients (25-28%) in water with 0.1% trifluoroacetic acid over varying gradient times (5, 12.5, 20 min) at 0.20 mL/min flow rate.

    • Main Results:

    • Observed experimental band compression factors for insulin were 0.56, 0.71, and 0.76 for gradient times of 5, 12.5, and 20 min, respectively.
    • These experimental values were 20-30% lower than theoretical predictions (0.79, 0.89, 0.93).
    • Improved agreement between experimental and theoretical values was achieved when accounting for the pressure gradient effect on local retention factor.

    Conclusions:

    • The experimental protocol provides accurate measurements of protein band compression in gradient RPLC.
    • Pressure gradients significantly impact band compression, especially for proteins, and should be included in theoretical models.
    • Gradient shape distortion due to finite acetonitrile retention is a factor for lower molecular weight compounds that requires theoretical consideration.